Key mechanism in DNA repair discovered

After a DNA molecules breaks, the broken ends search for an intact DNA region with the same sequence in order to get repaired. The image shows an artist impression of the contact point between a RecA-protein DNA molecule (the ‘broken end’; horizontal) and a DNA molecule (vertical), where it is probed whether both molecules have the same sequence. If they do not, they will break the contact. If the same sequence is found however, the molecules stably bind and the repair process is initiated. The present study discovered the mechanism of the recognition process from dual molecule experiments where individual DNA molecules can be manipulated with beads.

Scientists
from the Kavli Institute of Nanoscience at Delft University of
Technology have discovered a key element in the mechanism of DNA repair.
When the DNA double helix breaks, the broken end goes searching for the
similar sequence and uses that as a template for repair. Using a smart
new dual-molecule technique, the Delft group has now found out how the
DNA molecule is able to perform this search and recognition process in
such an efficient way. This week, the researchers report their findings
in Molecular Cell.

A staggering problem

Sometimes,
the DNA double helix gets broken: both strands are accidentally cut.
This presents a vital problem because cells cannot cope with such
damaged DNA. Genomic DNA instabilities such as these, are a known cause
of cancer. The good news is that an intricate DNA repair system exists
which is impressively error-proof and efficient. How does this work?

First,
proteins form a filamentous structure on the broken DNA end. Second,
this filament examines recently copied DNA or the second DNA chromosome
(remember that we have two copies of each chromosome) in search of a DNA
sequence that matches that of the broken end. Note that this is a
daunting task: given that, for example, our human genome contains three
billion base pairs, finding your few hundred base pairs of interest, is
really like finding a needle in a haystack.

'Still
this search process occurs within minutes and with great efficiency.
How that is achieved, has been a mystery for decades. The new
experiments from our group now resolve this by revealing the key step in
the process, the molecular recognition step', says scientist Iwijn de
Vlaminck, who was the postdoc that did the experiments in the group of
prof. Cees Dekker at Delft.

Search operation

'In
bacteria, the so-called RecA protein is responsible for conducting the
search operation. In E. coli bacteria, a filament of RecA protein formed
on DNA, searches and pairs a sequence within a second DNA molecule with
remarkable speed and fidelity. To do so, individual molecules of RecA
first come together to form a filamentous structure on the broken DNA.
The filament then grabs DNA molecules in its vicinity and compares their
sequence to the sequence of the broken DNA. When a sequence match is
found, both molecules bind tightly to one another allowing repair to
ensue', says De Vlaminck (since recently at Stanford University).

'We
found that the filament's secondary DNA-binding site interacts with a
single strand of the incoming double-stranded DNA during homology
sampling. Recognition is achieved upon binding of both strands of the
incoming DNA to each of two DNA-binding sites in the filament.'

The
data indicate that the fidelity of the search process is governed by
the distance between the DNA binding sites. The Delft experiments
clarify what exactly happens in the sequence comparison of the two
molecules, making clear why a 'wrong' sequence leads to quick
dissociation of the molecules while a 'correct' sequence makes a strong
bond leading up to further repair. These are the two elements that lead
to the impressive speed and high efficiency of the DNA repair process.

New instrument

The
team from TU Delft developed a unique new instrument that makes it
possible to independently manipulate an individual DNA molecule and an
individual RecA filament and to measure the strength of intermolecular
interactions. This dual-molecule manipulation instrument combines
magnetic-tweezer and laser-trapping-based DNA-molecule manipulation with
a laminar flow system. The setup also allowed to unwind the DNA helix a
bit, thus opening local regions where the normal duplex was
destabilized. This effect turned out to be crucial for the molecular
recognition process. Thus, the team was able to directly probe the
strength of the two-molecule interactions involved in search and
recognition and build a new model for it.